The goal of the proposed research is to develop efficient and widely applicable chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is one of the most common posttranslational modifications of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. However, a clear understanding of the structure-function relationships of glycoproteins is often hampered by their structural heterogeneity. Natural glycoproteins are usually produced as a mixture of glycoforms that are extremely difficult to isolate in pure form. To obtain homogeneous materials for structural and biological studies, we propose to systematically explore the trans-glycosylation activity of a special class of endoglycosidases, the endo-beta-N-acetylglucosaminidases (ENGases), for N-glycopeptide and N-glycoprotein synthesis. Some ENGases possess unique ability to transfer an intact oligosaccharide to a GlcNAc-containing peptide in a single step to form a new glycopeptide in a regio- and stereo-specific manner, thus providing a highly convergent route to glycopeptide synthesis. But the method suffers with serious problems such as the low yield in transglycosylation, the limitation of donor substrates, and the product hydrolysis. The proposed studies intend to solve these problems by exploring new substrates and screening new enzyme mutants. Three specific aims are proposed to achieve the goal. In specific aim 1, a range of oligosaccharide donor substrates, including sugar oxazolines (presumed transition state mimics) and p-nitrophenyl glycosides (ground-state but kinetically favorable substrates) of three major types of N-glycans will be synthesized and evaluated. The specific aim 2 is to discover ENGase mutants with new and/or enhanced transglycosylation activity by screening mutant library, using the coupled enzyme assay and the fluorescent resonance energy transfer (FRET)-based assay. Specific aim 3 focuses on total synthesis of several large and complex HIV-1 envelope glycoprotein fragments, as well as semi- synthesis of selected glycoproteins. In the long term, the proposed studies will contribute to the development of glycoprotein-based drugs. [unreadable] [unreadable] [unreadable]